Liver- and bone-derived isoenzymes of alkaline phosphatase in serum as determined by high-performance affinity chromatography.
To separate liver and bone alkaline phosphatase (ALP) isoenzymes in human serum, we used high-performance affinity chromatography (HPAC) on a column of wheat-germ lectin conjugated to 7-microns-diameter silica particles and an eluent containing N-acetyl-D-glucosamine (NAG). On-line spectrophotometric detection of ALP involved pumping diethanolamine-buffered p-nitrophenyl phosphate solution post-column. Bone and liver isoenzymes could be separated into two peaks with only 10% overlap when an exponential gradient was used. A linear-step gradient separated 80.9% of liver ALP and 91.6% of bone ALP in two distinct peaks. True bone and liver ALP peak areas for the linear-step gradient were determined by using correction factors, because each peak contained a co-eluted portion of the other ALP isoenzyme. The detection limit improved 10-fold over those of other techniques for ALP isoenzymes, owing to the relatively large sample that could be applied to the column. Correlation with a urea-inactivation procedure was reasonable for patients' serum samples (r = 0.98 and 0.79 for liver ALP and bone ALP, respectively).
Anderson
D J
DJ
Section of Clinical Chemistry, Mayo Clinic, Rochester, MN 55905.
Branum
E L
EL
O'Brien
J F
JF
D000469:Alkaline Phosphatase / Q000097:blood*; D001847:Bone Diseases / Q000175:diagnosis / Q000201:enzymology* / Q000378:metabolism; D001842:Bone and Bones / Q000201:enzymology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007527:Isoenzymes / Q000097:blood*; D008099:Liver / Q000201:enzymology; D008107:Liver Diseases / Q000175:diagnosis / Q000201:enzymology* / Q000378:metabolism
D007527:Isoenzymes; D000469:Alkaline Phosphatase